Cardiac G protein-coupled receptors (GPCRs) are important in regulating heart function. These GPCRs detect extracellular stimuli (agonists) and generate an intracellular signaling response crucial to controlling heart rate and contractility. Disordered signaling is observed in pathologies including congestive heart failure, cardiomyopathy and hypertension. ?2- adrenergic receptors (?2ARs) and M2 muscarinic acetylcholine receptors (M2Rs) are GPCRs that are co-expressed in cardiac muscle cells and operate to exert largely opposing modulatory effects. ?2ARs stimulate cardiac excitability primarily by coupling to the heterotrimeric G protein Gs. Conversely, M2Rs reduce excitability by coupling to Gi. Both GPCRs internalize after agonist- induced activation. Classically, internalized GPCRs are thought to be incapable of coupling to cognate G proteins. However, recent work has shown that some GPCRs, including ?2ARs, initiate a second phase of signaling by coupling to Gs on endosomes. It is presently not known if endosomal signaling is a feature of other GPCRs such as Gi coupled receptors and the significance of endosomal signaling to autonomic nervous system modulation of cardiac cells remains completely unexplored. The proposed work will test the hypothesis that ?2ARs and M2Rs are active in endosomes, and that these GPCRs continue to mediate opposing signaling effects after internalization. The proposed studies will first use HEK293 cells as an experimentally advantageous model system, in which endosomal Gs activation has been well established, and then examine cultured cardiac myocytes that more closely approximate the physiological environment of ?2AR and M2R operation in cardiac muscle. The specific aims of this project are to: 1) elucidate internalization behavior of M2R relative to ?2AR, 2) determine how M2R internalization impacts ?2AR signaling, and 3) test the relevance of endocytosis to ?2AR and M2R signaling in cultured cardiac myocytes. Both ?2ARs and M2Rs critically regulate cardiac function and do so through largely opposing cellular effects. The proposed aims will be addressed using a variety of techniques including microscopy, fluorescent flow cytometry, and quantitative PCR. The proposed studies investigate a fundamentally new aspect of cellular GPCR signaling, namely whether endocytosis facilitates functional coordination between these GPCRs or, alternatively, if endocytosis isolates a subset of ?2AR signaling actions from M2R modulation, which is fundamental to understanding the physiological relevance of cardiac endosomal signaling.